The plate culture method has been conventionally used for measurement of general live bacterial counts in foodstuffs, clinical samples or environments. However, the plate culture method requires time of about two days to obtain a result. Furthermore, with a bacteriological test based on culture using a generally used medium, it is difficult to detect bacteria injured in environments, bacteria injured by artificial stress (the former may be referred to as Viable-but-Non Culturable (VNC) cells, and the latter may be referred to as injured cells, in particular, in narrow senses) and so forth, and it has been desired to develop a quick and reliable method for counting live bacterium.
Flow cytometry (FCM) is a technique of flowing a sample in a flow cell at a constant flow rate to pass it through a laser beam, and measuring lights scattered by cells or other microparticles or fluorescence emitted by the same. Since it enables detection of microorganisms at a single cell level, it is used in recent years for detection of microorganisms not only in the fields of molecular biology and cell biology, but also for detection of microorganisms in environment, dairy products, drink, clinical specimens and so forth (for example, Patent documents 1 and 2, Non-patent documents 1 to 5).
However, FCM apparatuses (flow cytometers) used for this method are very expensive and have a large size, and they also requires skills for operation. Moreover, they still have problems to be improved or solved concerning economy, safety, simplicity, and reliability and actuality for distinction of live cells and dead cells of microorganisms for actual applications in the fields of foodstuffs, in which a wide variety of bacteria contaminate as non-injured bacteria, injured bacteria and dead bacteria.
For example, Patent document 1 discloses a method for detecting total bacteria from a liquid sample by using an ion-chelator, a protease, a detergent and a bacteriologically specific fluorescent dye. The ion-chelator, of which typical example is EDTA, must be used at a concentration of 1 to 5 mM, and if the concentration exceeds that level, cell walls and cell membranes of live bacteria of which cell walls are not injured may also be destroyed. The preferred concentration of the ion-chelator used in the method of Patent document 1 is about 6 to 17 mM, and therefore it has a problem that both dead bacteria and live bacteria are lysed. Moreover, the detection limit of this method is around 104 cfu/ml, and therefore if live bacterial count is low (103 cfu/ml or lower) in a liquid sample under a condition that only live bacteria exist in the liquid sample, the bacteria must be proliferated to a level of the aforementioned detection limit. Therefore, it cannot necessarily be considered a quick method.
Patent document 2 discloses a method of treating a body fluid sample with protease, lipase and nuclease, lysing leucocytes, thrombocytes and erythrocytes by ethidium bromide staining in a buffer comprising sodium borate, EDTA, formaldehyde and nonionic detergent (Triton X-100 etc.) to stain only bacteria with ethidium bromide, detecting and quantifying the bacteria based on fluorescence microscopy, flow cytometry, or the like. However, it is suggested that leucocytes and thrombocytes not lysed remain in the body fluid sample even after the protease, lipase and nuclease treatments, and live bacteria adsorb onto them to form complexes, and that both live bacteria and dead bacteria are stained, and thus it becomes difficult to determine whether bacteria are dead or alive. Furthermore, although Patent document 2 describes that the method is a method for detecting bacteria at a density as low as 10 cells/ml (sample) within a time of about 2 hours or shorter, often 45 minutes or shorter, it actually also discloses an example in which detection was not possible unless at least 104 cfu/ml or more of bacteria exist in a body fluid sample, and thus it is not suitable for detection of a small amount of microorganisms such as those in cow's milk.
Non-patent document 1 discloses a technique of utilizing a characteristic of SYTO63 that it penetrates cell walls and cell membranes of live cells and dead cells, and a characteristic of TO-PRO3 that it penetrates only cell walls and cell membranes of dead cells to attempt distinction of live bacteria and dead bacteria based on flow cytometry. In addition, it disclose an example in which live cells and dead cells were suspended in sterilized water, and distinction of live cells and dead cells was attempted in that environment. However, the dead cells were those boiled for 15 minutes, and the cell walls and cell membranes thereof were more highly injured compared with dead bacteria in actual foodstuffs. Therefore, this technique is a technique suitable only for dead bacteria in a limited range of foodstuffs such as cooked dishes, and conditions were not examined for ultra high temperature pasteurization, which is performed for cow's milk etc. and the latest foodstuffs, and kills only bacteria without denaturing proteins in foodstuffs.
Non-patent document 2 discloses a method of allowing proteinase K to act on UHT (ultra high temperature pasteurization) cow's milk to digest micellar casein, removing lipids by refrigerated centrifugation to detect bacteria in the cow's milk, and measuring total bacterial count (including live bacteria and dead bacteria) thereof, and a method of adding 0.1% Triton X-100 as a nonionic detergent to raw milk in addition to the aforementioned proteinase K to detect bacteria in the raw milk and measuring total bacterial count (count of live bacteria and dead bacteria). However, in the methods of Non-patent document 2, even if protease K is allowed to act on UHT cow's milk, micellar casein is not completely digested, and there are a lot of incomplete digestion products having a size comparable to those of bacteria. If a fluorescent nuclear stain agent such as SYTO BC or SYTO9 is made to act on such products, strong nonspecific adsorption occurs to make the distinction of them from live bacteria difficult. Moreover, it has also a problem that cell membranes of somatic cells such as bovine leucocytes and mammary epitheliocyte, considered as one of the contaminant milk components, are only slightly injured, and if they are subjected to staining with SYTO BC, SYTO9 or propidium iodide as they are, propidium iodide does not penetrate into them, and as a result, green fluorescence is emitted by chromosomal DNA to make distinction of the somatic cells from live bacteria difficult.
Non-patent document 3 discloses a method similar to the method of Patent document 1 except that the protease treatment is excluded as a method for measuring live bacterial count of lactic acid bacteria in yogurt or yogurt starter, and the method is described as a method of using a nonionic detergent and a chelating agent in combination. As the characteristic of the invention, it is described that the method enables destruction of somatic cells as contaminants and effective separation of fat globules. However, samples subjected to the aforementioned treatment contain a lot of contaminants originating in milk, and the detection limit for live lactic acid bacteria is degraded to a level as low as about 105 cfu/ml for yogurt or yogurt starter due to the contaminants. Therefore, the method requires extremely delicate determination of conditions for destroying only somatic cells and not injuring cell walls and cell membranes of live bacteria by adjusting the concentrations of the nonionic detergent and the chelating agent. Thus, the method is not suitable as a convenient and highly sensitive detection method for distinguishing live bacteria and dead bacteria.
Although ethidium monoazide (EMA, 8-azido-3-amino-6-phenyl-5-ethylphenanthradinium chloride) is generally known for the effect as an anticancer agent, it is a poison against topoisomerase II (type II topoisomerase) existing in mammalian cells (for example, Non-patent document 5). EMA disorderly intercalates into chromosomal DNAs, and then only intercalating EMA is converted into nitrene by irradiation of visible light, and binds to the chromosomal DNAs by covalent attachment. For example, by the action of topoisomerase, cancer cells adjust the helical degree of the DNA strands, or rewind DNA strands in order to perform replication of the DNA strands and gene expression (transcription of DNA), and the rewinding is achieved by cleavage of corresponding sites of the chromosomal DNAs and religation of the cleavage products. In this occasion, as for the function of EMA, the religation of DNAs by topoisomerase II is inhibited by the action of covalent attachment of nitrene derived from EMA at the time of the religation, and the fragmentation of the chromosomal DNAs is enhanced as a result. EMA not intercalating into DNA strands and existing in a free form is converted into hydroxylamine by visible light, but the hydroxylamine does not inhibit the activity of topoisomerase II.
As substances inhibiting such an activity of topoisomerase II, there are known, besides ethidium monoazide mentioned above, amsacrine, doxorubicin, ellipticine, etoposide, mitoxantrone, saintopin, and so forth. As substances inhibiting the activity of topoisomerase I, which has an activity similar to that of topoisomerase II, there are known camptothecin, topotecan, and so forth (for example, Non-patent document 6). Further, in the field of bacteria, as substances inhibiting the activity of bacterial DNA gyrase having an activity similar to those of the aforementioned enzymes, there are known ciprofloxacin, ofloxacin, enoxacin, pefloxacin, fleroxacin, norfloxacin, nalidixic acid, oxolinic acid, piromidic acid, and so forth (for example, Non-patent document 7).
However, there has not so far been reported at all use of these topoisomerase I poisons, topoisomerase II poisons, and bacterial DNA gyrase poisons for pretreatments of samples such as foodstuffs and clinical samples containing microorganisms in a test method for distinguishing live cells and dead cells of a microorganism for the purpose of realizing quick and highly sensitive detection.
As another method for detecting live bacteria, there has been proposed an automated system for conveniently and quickly detecting respiratory activity and esterase activity (Patent document 3). However, detection by this method is limited to a case where respiratory activity and esterase activity of the objective bacterium can be accurately measured.
As the state of microorganisms other than live bacteria, there are injured cells, VNC (Viable-but-Non Culturable) cells and dead cells. There is disclosed a method for detecting them by flow cytometry using cFDA (carboxyfluorescein diacetate), which emits green fluorescence in the presence of esterase, and propidium iodide (PI) (Non-patent document 8). However, this method is also a method that can clearly distinguish live cells, injured cells and dead cells only when the injury to cell walls of the injured cells comparatively advances. Therefore, when the injured cells are those showing a low degree of injury caused by low temperature long time pasteurization (LTLT) or high temperature short time pasteurization (HTST), or those showing a low degree of injury due to stress in environment, live cells and injured cells cannot be distinguished by this method.    Patent document 1: International Patent Application Unexamined Publication in Japan No. 9-510105    Patent document 2: Japanese Patent Publication (Kokoku) No. 6-55157    Patent document 3: Japanese Patent Laid-open No. 2002-281998    Non-patent document 1: Bokin Bobai, Vol. 31, No. 7, 2003, pp. 357-363    Non-patent document 2: Applied and Environmental Microbiology, Vol. 66, No. 3, 2000, pp. 1228-1232    Non-patent document 3: Applied and Environmental Microbiology, Vol. 68, No. 6, 2002, pp. 2934-2942    Non-patent document 4: Applied and Environmental Microbiology, Vol. 60, No. 12, 1994, pp. 4255-4262    Non-patent document 5: Biochemistry, Vol. 36, No. 50, 1997, pp. 15884-15891    Non-patent document 6: The Journal of Biological Chemistry, Vol. 270, No. 37, 1995, pp. 21429-21432    Non-patent document 7: The New England Journal of Medicine, Vol. 324, No. 6, 1991, pp. 384-394    Non-patent document 8: Applied and Environmental Microbiology, Vol. 68, 2002, pp. 5209-5216